Comparing modified Lee and White method against 20-minute whole blood clotting test as bedside coagulation screening test in snake envenomation victims

Abstract Background: Twenty-minute whole blood clotting test (20WBCT) and Modified Lee and White (MLW) method are the most routinely employed bedside tests for detecting coagulopathic snake envenomation. Our study compared the diagnostic utility of MLW and 20WBCT for snakebite victims at a tertiary care hospital in Central Kerala, South India. Methods: This single-center study recruited 267 patients admitted with snake bites. 20WBCT and MLW were performed simultaneously at admission along with the measurement of Prothrombin Time (PT). The diagnostic utility of 20WBCT and MLW was determined by comparing the sensitivity (Sn), specificity (Sp), positive and negative predictive values, likelihood ratios, and accuracy at admission with an INR value > 1.4. Results: Out of 267 patients, 20 (7.5%) patients had VICC. Amongst those who had venom-induced consumption coagulopathy (VICC), MLW was prolonged for 17 patients, (Sn 85% 95% confidence interval [CI]: 61.1-96.0) whereas 20WBCT was abnormal for 11 patients (Sn 55%, 95% CI: 32.04-76.17). MLW and 20WBCT were falsely positive for the same patient (Sp 99.6%, 95% CI: 97.4-99.9%). Conclusion: MLW is more sensitive than 20WBCT to detect coagulopathy at the bedside amongst snakebite victims. However, further studies are necessary for standardizing bedside coagulation tests in snakebite cases.


Background
Snakebite is a neglected disease of the poor population in India [1]. India is the largest contributor to the global burden of snakebite in terms of mortality with more than 50,000 deaths due to snakebite every year [2,3]. Mortality due to snakebite in India is mostly due to snake species that cause venominduced consumption coagulopathy (VICC), the most common species involved being the Russell's Viper (Daboia russelii) [3]. In the absence of a reliable bedside venom detection kit, clinicians rely on the patient's symptomatology, clinical signs, and laboratory detection of coagulopathy to determine the presence of envenomation and the need for snake antivenom (ASV) [4].
Various methods are available for bedside detection of coagulopathy, with the most commonly used being the twentyminute whole blood clotting test (20WBCT), modified Lee and White (MLW), and venous clotting time (VCT) [4]. These tests, which are modifications of Lee and White's original description of clotting time (LWCT), offer the potential advantages of being inexpensive, requiring limited resources, and being easily reproducible [4,5]. The limited availability of more standardizable conventional coagulation tests (CCT) such as prothrombin time (PT) with international normalized ratio (INR) and serum fibrinogen level, which are recognized for their ability to predict VICC at an early stage and with better sensitivity (Sn) and specificity (Sp), in most rural settings where snakebite is treated, can lead to delays in diagnosis [6,7].
The 20WBCT recommended by the World Health Organization (WHO) is the most routinely employed bedside screening tool for VICC assessment in India [8,9]. It is the tool of choice due to its cost-effectiveness in low and middle-income countries (LMIC) [4,7,10]. The 20WBCT has low Sn for detecting coagulopathy in snake envenomation compared to conventional coagulation tests like PT and Activated Partial Thromboplastin Time (aPTT) [7,11]. MLW is another test that has been employed for decades to assess coagulation in many hospitals in South India. It is an inexpensive bedside test used as part of the standard treatment protocol for snakebite victims [12]. We undertook the present study to determine the diagnostic utility of the MLW method in comparison with 20WBCT, at Emergency Department (ED) admission, amongst snakebite victims in central Kerala, Southern India.

Methods
This was a prospective observational unblinded diagnostic test study done among snakebite patients who presented to the ED of Jubilee Mission Medical College and Research Institute in South India during the study period (one year) after obtaining independent approval from the institutional review board and institutional ethics committee (01/19/IEC/JMMC&RI). The study was registered under clinicaltrials.gov (NCT03890016).

Participants
All patients with a 'history of snakebite' who presented to the ED during the study period were enrolled sequentially in the study after obtaining their consent. Patients with known bleeding disorders, those on anticoagulants/antiplatelets, individuals who received antivenom or blood products elsewhere, those with chronic liver disease/other hematologic disorders, and those who did not provide written informed consent were excluded from the study (Figure 1). All snakebite patients presenting to the ED were managed as per the institute's snakebite treatment protocol ( Figure 2).
History of snakebites, for the purpose of this study, was defined as 'patients or their bystander claiming in a sound mind and not under the influence of any inebriants, to have witnessed being bitten by a snake'.

Procedure
Phlebotomists who received training made sure to collect blood samples in accordance with the institution's protocol. They performed collections at admission, one hour, three hours, and six hours after the bite, and then continued to collect samples every six hours up to 24 hours in all cases where envenomation was suspected.
The samples collected were assessed for coagulopathy by two methods at the bedside; 20WBCT and MLW. 20WBCT and MLW were done simultaneously by a trained phlebotomist. 20WBCT works on the principle that blood clots on exposure to glass, whereas MLW in addition employs agitation of the sample by tilting the tube every minute, which speeds up the clotting process. Samples for doing PT were also collected at the same time.

20WBCT
To perform the 20WBCT, 2 ml of atraumatic venepuncture blood samples are collected in a new clean, dry, properly labeled, glass test tube (Borosilicate glass, 12*75mm) ( Figure 3). The blood sample is left undisturbed for a period of 20 minutes in this tube. At the end of 20 minutes, the sample is tilted approximately 60° to see if a stable clot has formed. A stable blood clot in a test tube for a 20WBCT test would appear as a solid mass at the bottom of the tube that maintains its shape and clings to the glass vessel without any breakage or dissolution on tilting the tube. The presence of a stable clot at the end of 20 minutes is noted as "normal" (Figure 4) and if no stable clot is formed; it is noted as "abnormal" ( Figure 5).

MLW
For MLW, 6 ml of atraumatic venepuncture blood sample is collected using a sterile plastic unlubricated syringe. Two ml each is dispensed into three new clean, dry, properly labeled, glass test tubes (Borosilicate, 12* 75mm), sequentially labeled T3, T2, and T1 respectively. The blood in T1 theoretically has the highest amount of Tissue Factor. The three test tubes are kept on a rack at ambient temperature. Although the emergency department and transfusion department where the study took place were air-conditioned, the temperature was not being monitored.
Using a stopwatch; timing is started as soon as the last test tube (T1) is filled. The test tube (T1) is left undisturbed for five minutes, following which the tube T1 is tilted approximately 60° to check for clot formation. This is repeated every minute while the other tubes are left undisturbed. After the blood in the T1 test tube has clotted, the T2 would be tilted every minute and examined. Following its clotting, T3 would be examined and the timer would be stopped at the time when a clot is noted in T3. The total time is documented in minutes as the CT value ( Figure 6). Once clotted, the sample in T3 would be further assessed every minute for up to 10 minutes to look for breakup or lysis of the clot. Breakage or dissolution of a formed clot if seen was noted as "clot lysis" (Figure 7). Absent clot formation at 20 minutes and clot lysis are both taken as prolonged MLW.

PT
Prothrombin time (PT) was done using an automated coagulation analyzer ACL-TOP 300, at admission for patients. Samples were collected in citrate tubes (Blue top, 3.2% sodium citrate) and centrifuged at the central lab. Platelet Poor Plasma was used for the test. Quality control was performed using the Pooled Normal Plasma daily as per the lab standards. Prolonged PT with an INR > 1.4 was taken as a surrogate standard for determining the presence of venom-induced consumption coagulopathy (VICC).

Sample size and statistical analysis
Based on the data abstracted for a pilot study in the same institute by Melit et al the sample size was calculated as 267 using the confidence interval set at 95% with z value at 1.96, with a sensitivity of 82%, 50% prevalence and precision of 7% [13].
We assessed the accuracy of the MLW and 20WBCT, with INR as the reference standard. Sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), likelihood ratios (LR+, LR-), and accuracy were calculated with 95% confidence intervals (CI).    The data were analyzed using IBM SPSS statistics 64-bit MS Windows ver. 25.0.0.0 (IBM Corp., Armonk, N.Y., USA). Descriptive statistics were performed to examine the relation between MLW and 20WBCT with simultaneously drawn PT values at admission, as the surrogate gold standard.

Results
About 326 patients were presented to the ED during the study period out of which 267 patients met the inclusion and exclusion criteria. Out of 267 patients, 20 (7.5%) patients had VICC. Amongst those who had VICC (n = 20), MLW was prolonged for 17 (85.0%) patients (Figure 8) whereas 20WBCT was abnormal for 11 (55.0%) patients (Figure 9). While doing MLW, 2 patients showed clot lysis i.e., MLW samples clotted at 18 minutes but when observed further showed dissolution of the formed clot after 4 and 6 minutes respectively. 20WBCT was recorded as normal for these patients while simultaneously reported PT was prolonged (INR > 1.4).       (Tables 3 and 4).

Discussion
In the absence of CCTs, bedside clotting tests represents a cost-effective and timely diagnostic test for clinicians to detect coagulopathy in snakebite victims, especially in a rural setting [4]. The 20WBCT introduced by Warrell et al. is a simple, relatively inexpensive, bedside test essentially a modification of the LWCT, routinely done in all snakebite patients in developing countries, especially in rural areas, where expensive tests like PT and APTT are often not available or affordable [9]. It is done to determine the presence of coagulopathy and also for the decision regarding the administration of ASV [8].
Although 20WBCT was intended for resource-limited settings, even hospitals capable of CCT (PT with INR, aPTT) use 20WBCT routinely to manage snake bite patients. The time to develop coagulopathy in individuals is dependent on multiple patient factors, the snake species, the amount of venom injected, and the time to the presentation at a hospital. This necessitates frequent sampling of the individual and the simplicity of taking a sample of blood in a glass tube, with no interventions, waiting for twenty minutes to check for a binary response as to which a clot is formed 'yes' or 'no', makes it a very cost-effective test. Although it is known to be specific, its sensitivity has been questioned in many studies. In an observational study from Sri Lanka of 140 cases of Russell's viper bite patients, the 20WBCT showed a sensitivity of 40% and specificity of 100%. The study concluded that 20WBCT was not sensitive enough to detect severe degrees of coagulopathy and the administration of antivenom was delayed [4]. In a subsequent study, they included trained research assistants and used glass tubes with standard dimensions thereby standardizing 20WBCT which improved the sensitivity to 82% [4,11]. The aggregate weighted sensitivity for 20WBCT at detecting prolonged PT at an INR > 1.4, from 12 studies (n = 2006) in a recent meta-analysis was 84% (95% CI 0.61 to 0.94) with a specificity of 91% (95% CI 0.76 to 0.97) [7]. Even though WBCT is the WHO -recommended bedside coagulation test to detect VICC, its low sensitivity and false negative results can lead to delayed administration of ASV in patients without clinical signs of envenomation. In our study, equally specific, MLW was more sensitive to detecting coagulopathy at admission in snakebite victims.
Results in this study were comparable to a recent study conducted in Brazil, MLW was studied in Bothrops sp. envenomation which showed moderate sensitivity (78%) for MLW [14]. MLW gives a quantitative value for clotting 'time' whereas 20WBCT gives a binary response as to whether a clot is formed 'yes' or 'no'. Although we initially hypothesized that serial MLW readings across multiple samples could result in a quantitative increase in time to clot as compared to 20WBCT aiding in early detection of coagulopathy, we were unable to demonstrate that, presumably because the study was underpowered for the same.
Snake venom is a cocktail of proteinaceous components that results in various hematotoxic effects ranging from "prothrombotic" to the "hyperfibrinolytic" spectrum of coagulation. 20WBCT looks at the prothrombotic end of the spectrum whereas MLW looks at both [15,16]. The MLW method continues to look for the stability of the clot and fibrinolysis in the form of clot lysis. MLW helps in the interpretation of coagulopathy from the same sample even if the 20WBCT shows a normal value at admission. Repeated/ delayed reading of 30 minutes proposed by Benjamin JM et al may circumvent this limitation in 20WBCT, however, this has not yet been replicated in other studies [17]. The evaluation and comparison of clotting time (MLW) with the clotting test (20WBCT) at different time frames, especially post-antivenom therapy, to detect normalization of coagulation and detecting secondary resumption of coagulopathy were beyond the scope of this study. Multiple tubes being used in MLW makes it more cumbersome for the novice, but eliminating the same, and employing the tilt and assessment for a single test tube and looking for clot lysis could combine the best of the two tests for a better bedside coagulation test, which may warrant further investigation.

Limitations
The blood sample collection and the tests were performed by trained lab assistants (phlebotomists) at admission. Having a trained phlebotomist perform the procedure would inevitably result in a proficiency bias affecting the generalisability of the result. Routine errors in performing 20WBCT and MLW leading to false positivity in the results like improper timing, improperly cleaned tubes, cleaning solution remnants, detergent coated tubes, improper sample collection, and procedure, etc. are avoided and the test would be more standardized which could also explain the high specificity noted in the results.
VICC was defined as prolonged PT (INR > 1.4) in this study and was not confirmed by other coagulation parameters like D-dimer and Fibrinogen levels. However previous studies support the use of INR values > 1.4 as a marker of VICC [1,6,7,17]. The study, although stringently recruited consecutive cases of snakebite victims, was unable to recruit adequate snakebite victims with coagulopathy in the set time frame from a single center.
The study follow-up of patients was limited to their time in ED (or up to 24 hours whichever was later) and there was a lack of details concerning responsible snake species. Mortality and morbidity outcome correlation were beyond the scope of our research question.
The time taken for both MLW and 20WBCT is 20-30 mins. The major difference is the personnel involved. This was not quantified in terms of operational expenses. The study did not undertake the identification of snakes because of insufficient funding for specimen storage. Additionally, there was a valid concern that in the absence of antivenom or snake venom detection kits in the region, human-wildlife interaction could occur, leading to no change in patient treatment outcomes but potentially resulting in unnecessary snake killings. Nevertheless, it is the authors' firm belief that a comprehensive understanding of the local snakes is essential to comprehend the distinctions in the clinical manifestation of hematotoxic snakebites in the region. Future research should include species identification, larger sample sizes to account for venomous bites, and a costeffectiveness analysis to assess the economic impact of the test.

Conclusion
In a setting with standardized training, the MLW test is more sensitive in detecting VICC when compared to 20WBCT amongst snakebite patients. However, to consolidate these findings, further studies involving larger sample sizes, multiple snakebite treatment centers, and a substantial number of envenomed patients are necessary. Additionally, extending the interpretation time of the 20WBCT through continued reading of the blood sample, along with exploring comprehensive analysis of additional hematological parameters, holds potential for improving the study's robustness. Our ongoing efforts aim to validate these results and provide a more comprehensive understanding of this subject [18].

Ethics approval
The study was approved by the institutional review board and the institutional ethic committee of Jubilee Mission Medical College and Research Institute (01/19/IEC/JMMC&RI). The study was registered under clinicaltrials.gov (NCT03890016).